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Aims
Consider the biological context of cell division;
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What happens if it goes wrong?
Describe the key events of the cell division cycle in eukaryotic organisms
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DNA replication
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Describe the key features of a plasmid cloning vector and explain their role in the cloning
process
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coli)
Most eukaryotes employ a more complex mechanism
Mitotic Cell Cycle
o G1 Phase – Cells double in size and prepare for DNA replication
o S Phase – DNA replication begins at origins of replication and continues until all DNA
has completed a single round of replication
o G2 Phase – Second growth phase; this is where all organelles are duplicated and the
cell prepares for mitosis
o M Phase – Mitosis; a segregation of sister chromatids to form two identical sets of
chromosomes
o C Phase – Cytokinesis; two new cells are formed, each carrying an identical set of
chromosomes
Interphase: The Key Elements;
o Chromosomes are relatively diffuse, cannot be seen individually
o Nuclear Envelopes are the double membrane that separates the nuclear contents
from the cytoplasm
o Microtubules (MTs) are like dynamic scaffolding poles, form one component of the
cytoskeleton, and these MTs extend from MT Organising Centres
o Centrosomes are the major MTOCs of most animal cells
Interphase: G1:
In terms of chromosomes, this is before DNA replication, therefore there is only one
chromatid per chromosome, the NE is intact, and MTs are originating
Interphase: S
DNA is replicating from the origins of replication, NE still intact, MTs continuing to originate
from a now duplicated Centrosome
Interphase: G2:
DNA replication is complete, two identical sister chromatids per chromosome, the NE is
intact, MTs still originating, but the centrosomes are separating towards the end of G2

Mitosis
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Prophase – chromatin condense, chromosomes becoming visible, NE breaks down
Metaphase – chromosomes are arranged at central plane, two chromatids per
chromosomes
Anaphase – Sister chromatids segregated to opposite poles of the cell, now single
chromatids
Telophase – new nuclei reform, chromatic de-condenses, and interphase cytoskeleton is
established

Cell cycle control
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Errors in the cell cycle can be catastrophic for the cell and for the whole organism
In multicellular organisms, only relatively few cells retain the ability to divide
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CDK activity only occurs when it is complexed with a cyclin

Sex and Meiosis
Ploidy
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In sexually reproducing organisms, the number of chromosomes in each somatic cell is
double that seen in their gametes
The basic number of chromosomes seen in the gametes is called the haploid number
(because it’s half, get it?)
The somatic cells which contain two copies of each chromosome are referred to as diploid
(double)

Sexual reproduction
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Lifecycles that allow recombination of genomes by alternation between haploid and diploid
states are called sexual lifecycles
The lifecycle of a human being is largely spent in the diploid state but this is not true for all
sexually reproducing organisms
Yeast are largely haploid, mosses are both

Meiosis
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In contrast to mitosis, meiosis involved two successive divisions, and is preceded by one
round of chromatid replication
The first is a reduction division, that halves the chromosome number from diploid to haploid
The second meiotic division is an equational division which halves the number of chromatids
per chromosome
As in mitosis, both meiotic divisions have four phases;
Prophase, Metaphase, Anaphase, Telophase
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from mitosis)
And the second meiotic division (similar to mitosis)

Prophase 1 (Meiosis)
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EARLY: Chromatin condenses, chromosomes pair up, and are “zipped” up by SC
MID: once pairing up completes, crossing over happens, causing reciprocal exchange
between homologues
LATE: the SC breaks down, chiasmata is a thing at this point

Metaphase 1

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Chromosome pairs are aligned at the central plane of the cell by spindle microtubules (SM)
Orientation of homologous pairs is random

Anaphase 1
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SM pull homologues apart
Chiasmata resolve towards the ends of the chromosomes
At the end, homologous pairs have segregated

Telophase 1
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At the end of meiosis 1, the original diploid cell nucleus has given rise to two haploid nuclei
However, the chromosomes still have two chromatids and must go through second division
to complete the process

Metaphase 2
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V similar to mitosis, the SM align al chromosomes at the central plane

Anaphase 2
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Sister chromatids are segregated, if crossing over has occurred, they’re no longer identical
Ploidy is not changed, but each chromosome has only a single chromatid

Telophase 2
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At telophase 2, nuclear envelopes reform around the four haploid groups of chromosomes
and the chromatin decondenses

Sex Determination
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Most animal species and many plant species have distinct male and female forms
Some reptiles and fish show environmental sex determination
But genetic sex determination mechanisms are more common
The simplest mechanism for genetic sex determination is found in some mosquitoes
The sex is determined by a single gene locus “M” (Mm = male, mm = female)
Note that this system automatically generates equal numbers of each sex

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In many organisms, the two sexes have different chromosome complements – Karyotypes
Most of the chromosomes occur in pairs, and are physically indistinguishable from their
partners – autosomes
One pair of chromosomes are often visibly different from each other – sex chromosomes
How do sex chromosomes determine sex in mammals? All gametes carry one X, half will
have another X and the rest will have a Y
During meiosis in females, errors in sex chromosome segregation can lead to abnormal
gametes
A small number of phenotypic males have an XX genotype & vice versa
Shown to be due to the transfer of a small region of the Y chromosome to the X
This region contains a single gene that has since been shown to confer maleness (the SRY
gene)
In mice, the transfer of the SRY gene to an autosome by genetic manipulation results in XX
males, phenotypically normal, but they’re sterile

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In Drosophila, sex is determined by the ratio of X chromosomes to sets of autosomes
Birds are like mammals, but the female is heterogametic
Grasshoppers are like Drosophila, but with no Y chromosome
Although sex chromosomes determine sex, they don’t carry all the genes necessary for
determining the sexual phenotype
They also carry many other genes which have nothing to do with sexual development
Genes carried on sex chromosomes are said to be sex linked and show unusual patterns of
inheritance
In mammals, X & Y only share a short region of homology, which is why there are gene loci
on X and not on Y
So males only carry one allele of most the genes that are on X
Males inherit their Y from their father, and X from mother
Males are HEMIZYGOUS for X-linked genes, hence recessive mutant allele s of X-linked genes
will always show in males
Male Drosophila have only 1 copy of most X-linked genes, therefore female flies have twice
the capacity to synthesise X-linked gene products
Drosophila males compensate by doubling levels of gene expression from their X
In female mammals, only one X is active in each cell
The inactive X is highly condensed & called a Barr Body

Transcription
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DNA is the master copy
Transcription acts to amplify the info
It can be regulated with gene expression
Transcription is performed by a multi-subunit enzyme called RNA polymerase
Eukaryotes have three types of RNA polymerase, Prokaryotes like E
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0001% of the
cell’s DNA
This makes the physical analysis of a specific gene difficult
Restriction Endonucleases are used to cut DNA at specific sequences
They’re produced by bacteria as a defence against viral infection
Many different Res are now commercially available

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E
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coli)
Most eukaryotes have several (cats have 38)
Individual eukaryotic chromosomes contain either:
A single DNA molecule prior to DNA replication
Or two identical DNA molecules following DNA replication

Eukaryotic Chromosomes
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They contain linear DNA molecules
This is an SEM of a typical chromosome at metaphase of mitosis
This is following DNA replication
o This means it has two sister chromatids
o Each will contain an identical DNA molecule
Sister chromatids are physically joined at a region called a centromere, this is often seen as a
constriction along the length of the chromosome
Chromosomes can be classified according to the position of their centromere:
o Near the centre = metacentric
o Towards one end = acrocentric
o Right at one end = telocentric
The centromere holds sister chromatids together and is the site of kinetochore formation
during mitosis and meiosis
Kinetochores form during prophase on either side of the centromere, they capture the
positive ends of microtubules, thus connecting the chromosome to the poles of the mitotic
spindle
The ends of the chromatids are called telomeres
Telomeres have an important structure, as they contain the free ends of the DNA molecule
Telomeres contain the free ends of the chromosomal DNA molecules

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This is a problem for the cell for two reasons:
o The normal mechanism of DNA replication cannot extend all the way to the end of a
linear chromosome
o The free end of a chromosome looks very similar to the end of a broken DNA
molecule

Telomere Maintenance
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Since the normal mechanism of DNA cannot extend all the way to the end of a linear
chromosome, an alternative mechanism has evolved to prevent chromosomes getting
shorter every time a cell divides
A special DNA polymerase called telomerase adds simple sequence repeats to maintain the
telomeres
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Garrod reasoned that people who suffer from alkaptonuria are unable to metabolise
homogentisic acid
Garrod examined family pedigrees and deduced that affected individuals are homozygous
for a recessive mutant allele of a single gene
He concluded that alkaptonuria is a genetic disease caused by the absence of an enzyme
necessary to metabolise homogentisic acid
Garrod’s insight wasn’t supported experimentally until 1941, when Beadle and Tatum
published the “One Gene – One Enzyme” hypothesis
Their Nobel prize work exploited the genetic analysis of a fungus Neurospora crassa
Neurospora is normally haploid and reduces mitotically by the production of asexual spores
called conidia

Wild type Neurospora are prototrophic, they are able to synthesise all the molecules they need,
given sufficient energy and minerals
Mutant Neurospora are unable to synthesise specific metabolites are called auxotrophs

The one gene – one enzyme Hypothesis
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A gene controls the production and/or the activity of a single enzyme, for example:
The arg+ gene must encode an enzyme involved in the synthesis of arginine
Modern interpretation = “a gene encodes the production of a single polypeptide”

Metabolic Pathways
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Beadle and Tatum isolated lots of auxotrophic mutants, in particular, they found 12 which
were all unable to synthesise arginine, the question is; were all these arg- strains mutant for
the same gene, or is more than one gene required for arginine synthesis?
To answer this, we must consider a cross between arg1 and arg2, if they’re mutations of the
same gene, all the progeny will be arg auxotrophs
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FT inserts a sugar (H-antigen) onto a protein in the plasma membrane of red blood cells
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Individuals homozygous for a null allele of the H gene (hh) are very rare
The hh genotype prevents the expression of the ABO phenotype
All hh people are blood typeO, regardless of their genotype at the l gene locus
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However, even when all those conditions are fulfilled, if the gene products do not function
independently, the phenotypic ratio may be different



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